In vivo observations of cell trafficking in allotransplanted vascularized skin flaps and conventional skin grafts

Abstract

The problem of allogeneic skin rejection is a major limitation to more widespread application of clinical composite tissue allotransplantation (CTA). Previous research examining skin rejection has mainly studied rejection of conventional skin grafts (CSG) using standard histological techniques. The aim of this study was to objectively assess if there were differences in the immune response to CSG and primarily vascularized skin in composite tissue allotransplants (SCTT) using in vivo techniques in order to gain new insights in to the immune response to skin allotransplants. CSG and SCTT were transplanted from standard Lewis (LEW) ad Wistar Furth (WF) to recipient transgenic green fluorescent Lewis rats (LEW-GFP). In vivo confocal microscopy was used to observe cell trafficking within skin of the transplants. In addition, immunohistochemical staining was performed on skin biopsies to reveal possible expression of class II major histocompatibility antigens. A difference was observed in the immune response to SCTT compared to CSG. SCTT had a greater density cellular infiltrate than CSG (p<0.03) that was focused more at the center of the transplant (p<0.05) than at the edges, likely due to the immediate vascularization of the skin. Recipient dendritic cells were only observed in rejecting SCTT, not CSG. Furthermore, dermal endothelial class II MHC expression was only observed in allogeneic SCTT. The immune response in both SCTT and CSG was focused on targets in the dermis, with infiltrating cells clustering around hair follicles (CSG and SCTT; p<0.01) and blood vessels (SCTT; p<0.01) in allogeneic transplants. This study suggests that there are significant differences between rejection of SCTT and CSG that may limit the relevance of much of the historical data on skin graft rejection when applied to composite tissue allotransplantation. Furthermore, the use of novel in vivo techniques identified characteristics of the immune response to allograft skin not previously described, which may be useful in directing future approaches to overcoming allograft skin rejection.

Figure 1

Isolation, placement and imaging of skin within a composite tissue transplant. The skin supplied by the epigastric vessels and gastrocnemius muscle were isolated on the common femoral vessels (1a). The flap was depiliated with Nair^® hair removing ointment prior to isolation, whereas the recipient groin site was only shaved, facilitating differentiation of donor and recipient tissues on imaging (1b). The skin flap underwent in vivo confocal imaging (1c) initially daily with the animal under sedation.

Figure 2

Representative examples of recipient cell influx into skin flaps and skin grafts transplanted across isogeneic and allogeneic barriers. There was no significant difference in the numbers of cells observed at the depth of greatest cell density at the center and edge of CSG at any time-point (isogenic CSG (2c) p > 0.1; allogeneic CSG (2d) p > 0.1). In contrast, there were significantly more cells in the highly vascularized center of SCTT than either the skin flap edge (isogeneic SCTT (2a) p < 0.05; allogeneic SCTT (2b) p < 0.05) or the center of comparable isogeneic (Figure 2a cf. 2c; p < 0.03) or allogeneic (2b cf. 2d; p < 0.01)CSG. There was greater infiltrate in to both the center and edge of rejecting allogeneic than isogeneic SCTT (2b cf. 2a; p < 0.01) and CSG (2d cf. 2c; p < 0.02).

Figure 3

Endothelial class II expression on vasculature of rejecting skin. MHC Class II expression was observed on immunohistochemical staining of biopsies taken from allogeneic SCTT taken 4 days after transplantation (3a), but not on isogeneic SCTT (3b) (×40 magnification).

Figure 4

Recipient cell types within transplanted skin Infilitrating recipient cells included dendritic cells, which were observable on confocal microscopy in allogeneic flaps from two days after transplantation (4a), but not observable at any time-point in CSG (4b). Both CD8+ (4c) and Class II positive (4d) lymphocytes were identified in allogeneic and isogeneic CSG and SCTT on immunohistochemical staining (×40 magnification).

Figure 5

Clustering of infiltrating cells observed around blood vessels and hair follicles in allogeneic skin transplants. Clustering of infiltrating cells was observed around blood vessels (5a; vessels colored red, arrows indicating perivascular cell clustering; p < 0.01) and hair follicles (5b; arrows indicating cells pallisading around hair base; p < 0.01) in allogeneic SCTT, and around hair follicles in allogeneic CSG (5c; arrows indicating cells pallisading around hair base; p < 0.01), but not in isogeneic SCTT (p > 0.1) and CSG (p > 0.1), 4 days after transplantation. Adequate Evans blue dye injection to image CSG vessels was confirmed by good vessel detection in the ear (5d). However, few vessels were visible in CSG 4 days after transplantation, preventing assessment of cell clustering around CSG vessels.